Malaria is still a devastating infectious disease of the world. Approximately 300 - 500 million people become infected yearly, with relatively high rates of morbidity and mortality. In fact, the WHO estimates that 2 - 3 million children die of malaria in Africa alone, every year. The overall aim of this proposal is to optimize adenoviral (Ad) vector in order to elicit a robust anti-malarial immunity as an effective malaria vaccine. Our earlier studies have shown that a recombinant Ad expressing a major malaria antigen, circumsporozoite (CS) antigen of Plasmodium yoelii, AdPyCS, could induce a protective anti-malarial immunity, which is mediated by CD8+ T cells. However, AdPyCS was unable to induce high levels of antibody response against malaria parasites. Because Ad is known to elicit a strong pre-existing anti-Ad immunity that is primarily against its capsid proteins, we hypothesized that the insertion of a foreign epitope into the capsid proteins results in an induction of a robust epitope-specific humoral response. To test this hypothesis, we have inserted an immunodominant B cell epitope of the PyCS protein into two different capsid proteins, i.e. one in the fiber and another in the hexon, of Ad that expresses an entire PyCS protein fused to GFP, as a transgene. After multiple immunization of each capsid-modified, or unmodified Ad(PyCS+GFP), we found that Ad(PyCS+GFP) expressing the B epitope in the fiber, Ad(PyCS+GFP)-F/B, induces a highest level of protective anti-malarial immunity, as well as anti-malarial humoral response. In Aim 1 of this proposal, we plan to seek the reasons why Ad(PyCS+GFP)-F/B can induce a strongest anti-malarial protection than other Ad-based vaccines. We will first confirm the protective capacity of Ad(PyCS+GFP)-F/B and then determine the levels of malaria-specific humoral and cellular responses induced by the different Ad vectors and characterize their responses in detail. Our next hypothesis is that a repeated immunization or a pre-existing immunity still affects capsid-modified Ad, reducing the production of the PyCS protein that includes a CD4+ epitope from the transgene. In Aim 2, therefore, we will construct a novel Ad vector by inserting a malaria-specific CD4+ epitope into various capsids of Ad(PyCS+GFP)-F/B, and determine whether the insertion of a CS-specific CD4+ epitope into the Ad capsid protein could elicit a strong CS-specific CD4+ T cell (helper) response, thereby enhancing further a robust anti-CS antibody response. It is possible that capsid modification of Ad makes the virus escape from anti-capsid antibodies, and that capsid-modified Ad is able to maintain its infectivity in vivo. In the last Aim, therefore, we will determine the tissue/cellular tropism and the intracellular trafficking of capsid-modified Ad and compare to those of unmodified Ad. PUBLIC HEALTH RELEVANCE: Malaria is still a devastating disease. The aim of this project is to develop a new adenovirus- based malaria vaccine that can elicit not only a strong malaria-specific cellular response, but also a robust malaria-specific humoral response. The simultaneous induction of high levels of both arms of the immune responses should lead to a successful induction of a potent protective immunity against malaria, thus ultimately placing Ad-based malaria vaccine, as a promising vaccine.